Rates of antibiotic resistance among pathogenic bacteria have risen to alarming levels. New strategies and alternatives to traditional antibiotics are needed to combat this health threat and derail the evolutionary arms race leading to resistance. One such alternative is the use of anti-virulence therapeutics. Rather than disrupting essential biological processes as for conventional antibiotics, anti-virulence approaches target bacterial systems that are only required to cause disease within the host. Thus, there should be less pressure for the development of resistance. In addition, anti-virulence strategies avoid the detrimental side effects of broad-spectrum antibiotics on the normal bacterial flora. Toward the goal of developing novel alternative therapeutics, we have discovered that the small molecule nitazoxanide (NTZ) inhibits pilus biogenesis by the conserved chaperone/usher pathway in Gram-negative pathogenic bacteria. Pili (fimbriae) are virulence- associated surface structures that mediate adhesion to host cells and colonization of host tissues. Pilus- mediated adhesion is critical for early stages of infection, allowing the bacteria to establish a foothold within the host. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where fluid flow washes away non-adherent pathogens. Following bacterial attachment, pili also modulate host cell signaling pathways, promote or inhibit invasion inside host cells, and mediate bacterial- bacterial interactions leading to formation of community structures such as biofilms. Pili thus function at the host-pathogen interface both to initiate and sustain infection, and represent attractive therapeutic targets. We have found that NTZ inhibits pilus assembly in uropathogenic as well as diarrheagenic strains of Escherichia coli. Moreover, we have determined that the inhibitory effect of NTZ is due to specific interference with proper maturation of the usher protei in the outer membrane. The usher provides the pilus assembly and secretion platform and is essential for pilus biogenesis. This proposal will test the hypothesis that NTZ targets the machinery required for insertion of the usher protein in the outer membrane, and that NTZ analogs will function as potent and specific inhibitors of pilus biogenesis by the CU pathway. The specific aims of this study are to: 1) determine the mechanism of action by which NTZ inhibits pilus biogenesis; 2) identify and characterize the direct target of NTZ; 3) develop and test NTZ-based derivatives with improved potency and pharmacological properties; and 4) test optimized compounds in cell culture and animal models of infection, focusing on uropathogenic E. coli, but also testing Klebsiella pneumoniae. The novel pilicide compounds developed by this proposal will represent a new class of anti-infective agents that target virulence factor secretion and the assembly of virulence-associated surface structures in multiple Gram-negative antibiotic threat pathogens.